Reverse progeny mapping

ABSTRACT

Provided is a method for mapping traits in organisms, in particular in plants. The method comprises a) providing a population of SDR-0 organisms, in particular plants, that each arise from one member of a population of unreduced cells resulting from second division restitution, in particular a population of unreduced spores; b) producing SDR-1 progeny populations of each of these SDR-0 organisms; c) phenotyping the SDR-1 progeny populations to identify segregating traits within each SDR-1 progeny population; d) if segregating progeny are present in a SDR-1 progeny population, genotyping the corresponding SDR-0 organism and comparing the genotype thereof with the genotype of the other SDR-0 organisms to identify heterozygous chromosomal regions associated with the occurrence of the segregating trait identified in the SDR-1 progeny population.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/848,800filed Aug. 31, 2007, now U.S. Pat. No. 7,860,658, which is acontinuation-in-part of International application No. PCT/EP2006/002096,filed Mar. 2, 2006, published as WO 2006/094774 on Sep. 14, 2006, andclaiming priority to EP 05075519.8, filed Mar. 3, 2005 and EP06075041.1, filed Jan. 6, 2006.

All of the foregoing applications, as well as all documents cited in theforegoing applications (“application documents”) and all documents citedor referenced in the application documents are incorporated herein byreference. Also, all documents cited in this application (“herein-citeddocuments”) and all documents cited or referenced in herein-citeddocuments are incorporated herein by reference. In addition, anymanufacturer's instructions or catalogues for any products cited ormentioned in each of the application documents or herein-cited documentsare incorporated by reference. Documents incorporated by reference intothis text or any teachings therein can be used in the practice of thisinvention. Documents incorporated by reference into this text are notadmitted to be prior art.

FIELD OF THE INVENTION

The present invention relates to a method for mapping traits inorganisms, in particular in plants.

BACKGROUND OF THE INVENTION

Complex crop traits such as yield, stress tolerance, metabolitecomposition and related phenomena such as heterosis and combiningability are difficult to study due to their quantitative genetic natureand strong interaction with the environment. In addition, the geneticsof such traits/phenomena caused by them is very often also complex andmostly quantitative and polygenic, which means that the resultingphenotype is caused by the interaction of the different alleles that areencoded by different genetic loci.

Attempts to characterise the individual loci that contribute to aquantitative trait have been successful when each of the individual locihas a measurable contribution to the total effect irrespective of thepresence or absence of alleles of the other loci, which contribute tothe quantitative trait. In this case the individual QTLs as they arecalled are of an additive nature and inherit in a simple Mendelianfashion.

Several methods for QTL mapping have been extensively described, howevermost of these methods fail when phenotypes are caused by the interactionof numerous heterozygous loci, especially when such loci areinterdependent. This means that two or more specific loci need to bepresent simultaneously for the expression of a specific trait. In theabsence of the required alleles on either of the two loci the phenotypictrait will not be expressed. The individually required alleles can occureither in homozygous or heterozygous form. Depending on the specifictrait, different genetic constitutions of the loci may be required. Forinstance a measurable effect is only observed when 2 or more loci arepresent in heterozygous state and no effect is observed when eitherlocus is homozygous. In such a case, one could state that such loci areinterdependent.

As mentioned before, complex traits such as yield and stress tolerance,are of high industrial importance, and therefore, it is highly desirableto have tools like molecular markers linked to these complex traits,which allow for increased efficiency of breeding for such traits indifferent crops.

Contemporary plant breeding is routinely using genetic (molecular)marker technologies such as AFLP, RAPD's, SSR's, SNP's etc, for a reviewsee e.g. Lakshmikumaran, T. et al., Molecular markers in improvement ofwheat and Brassica. In: Plant Breeding—Mendelian to Molecularapproaches. H. Jain and M. Kharkwal (eds.) Copyright 2004 NarosaPublishing House, New Delhi, India, page 229-255.

Molecular markers are very desirable as diagnostic tools that indicatethe presence of a particular trait even in a developmental stage duringwhich the trait is not expressed. In addition, molecular markers areinsensitive to environmental conditions.

As an example, molecular markers (for example in the form of SNP=singlenucleotide polymorphism, or associated with DNA bands on agarose orpolyacrylamide gels) can be found that are genetically linked to genesthat are responsible for the colour of pepper fruits when they are ripe.A DNA sample taken from a seedling can be used to determine which colourthe fruits of the plant will eventually have. So in this case there is adirect association between the presence of a particular DNA sequencethat is being “called” and the presence of a particular trait.

In essence, the same procedure is true for many polygenic traits (seee.g. Tanksley S., Mapping polygenes, Annu. Rev. Genet. 1993, 27:205-233). In the latter case, the trait, whatever it may be, forinstance disease resistance, resistance to stress, production ofvitamins etc., may be controlled by more than one locus. It is assumedthat the contribution of every individual locus, and its associated DNAmarker can be measured and that the sum of the different loci and theirrespective DNA markers will phenotypically result in the presence of theparticular trait (to some extent). This concept traces back to theclassical work of R. A. Fisher (The correlations between relatives onthe supposition of Mendelian inheritance, Trans. R. Soc. Edinb. (1918)52, 399-433), who linked Mendelian genetics with earlier statisticalapproaches of correlation between relatives, to explain quantitativelyinherited traits.

Eukaryote chromosome mapping by recombination is a well know techniquefor the person skilled in the art (Griffiths A J F et al., (2005)Eukaryote chromosome mapping by recombination, In: Introduction toGenetic Analysis, 8th edition. W. H. Freeman and Company, New York p115-137).

The mapping of segregating traits, i.e. QTL-mapping (QTL=QuantitativeTrait Locus), is not solely dependent on technical issues orrecombination but equally important is the accurate observation orscoring, qualitatively or quantitatively, respectively, of thephenotype. In this respect, when mapping complex traits or effects, theperson skilled in the art is preferentially using a population ofdoubled haploid lines (DH) or a population of recombinant inbred lines(RIL), which are segregating for the trait(s) of interest, and which arederived from a single F1 plant.

DH-lines are derived directly from the haploid F1-plant gametes, byplant regeneration and chromosome doubling. RILs are highly inbredlines, derived by single seed descent (SSD), i.e. via inbreeding overseveral generations, where each individual plant provides one seed forthe next generation, starting in the F2.

Alternatively, so called Near Isogenic Lines (NIL) are used. NILs arehomozygous lines that differ for a small DNA fragment. They are usuallyderived from backcrosses, but can also be obtained from segregating RILs(Tuinstra et al., (1997) Theor. Appl. Genet. 95: 1005-1011).

DH-lines, RILs and NILs greatly contributed to contemporary genetics andgenetic mapping. The advantage of such pure lines exactly lies in thefact that phenotypic variation between lines (inter-line variation) iseasily recorded as compared to segregation at the level of individualplants for a classical F2 mapping population. The availability of purelines is of course increasingly important as also environmentalinfluence may be accounted for by replication of genetically identicalplants of a pure line. This in contrast to single, unreplicated,individual F2-plant phenotypes that are the product of the interactionof genes and environment.

The elucidation of complex effects such as heterosis or combiningability between lines is one of the biggest challenges for contemporarygenetics and plant breeding. For heterosis, several hypotheses have beenformulated (see e.g. Birchler J et al. (2003) The plant Cell 15,2236-2239). The so-called historical explanations for heterosis are“overdominance” and dominance. Overdominance refers to the idea thatallelic interactions occur in the hybrid such that the heterozygousclass performs better than either homozygous class. Dominance refers tothe situation in which the suboptimal recessive allele of one parent iscomplemented by the dominant allele of the other parent. Whereasheterotic effects explained by dominance can in principle be fixed in ahomozygous state, it is obvious that for effects explained byoverdominance this is impossible. It has recently become clear that thetwo competing single-locus explanations for heterosis are insufficientand that also epistatic effects, i.e. inter-locus interactions, play amajor role as the genetic basis of heterosis (Yu S B et al. (1997) Proc.Natl. Acad. Sci. USA 94: 9226-9231).

As mentioned before, traditionally used mapping population structureswith homozygous individuals, such as Recombinant Inbred Line populations(RILs) and Doubled Haploid (DH) populations, cannot easily be appliedfor mapping the specific effect of the heterozygous state at a certainlocus. This disadvantage has been overcome by crossing these populationswith testers, and assessing the phenotypes of the offspring hybrids.However, this approach has three disadvantages. First, it requiresadditional labour, space and time. Furthermore, it compares theheterozygous state of a locus with only one of the two possiblehomozygous states, unless at least one additional tester is used. Andfinally, it does not fully assess the interaction between theheterozygous locus and the genetic background, i.e. gene interactionwith specific effects due to heterozygosity.

The use of diallel mating populations, as proposed by Charcosset et al.(1994) and Rebaï et al. (1994) (both in: Biometrics in plant breeding:applications of molecular markers; Eds: Ooijen J. and Jansen J.CPRO-DLO, Wageningen, The Netherlands), overcomes part of the latter twodisadvantages, but requires even more labour and space.

F2- and back-cross populations can be applied to assess for mapping thespecific effect of the heterozygous state at a certain locus. However,only limited gene interaction is allowed in the F2-based QTL-analysis,because of the available parameter space in the statistical model, whichis limited by population size. Backcross populations require more timeand labour to produce them, and the effect of the heterozygous state ata certain locus is only estimated for the genetic background of therecurrent parent, without taking into account possible interactions withother loci.

Another approach to avoid large investments in time, space and labour todevelop mapping populations is linkage disequilibrium mapping(LD-mapping; Kraakman A T W et al. (2004) Genetics 168, 435-446; Kraft Tet al. (2000) Theor. Appl. Genet. 101, 323-326). This method makes useof available existing genetic material, such as varieties and genebankaccessions. If this material is sufficiently heterozygous, for example amapping set of hybrid varieties, it is possible to estimate the specificeffect of the heterozygote loci. However, in general LD-mapping methodsdo not consider epistatic effects and require large numbers ofaccessions to detect additively working QTLs within the statisticalnoise caused by the epistatic effects, which are due to the differentgenetic backgrounds across all accessions. (Flint-Garcia S A et al.(2003) Annu. Rev. Plant Biol. 54, 357-374).

Traits that are dependent on the combination of the allelic constitutionof two or more loci are much more difficult to identify or map. Inpopulation genetics this interaction between several loci is called‘epistasis’. In this case the contribution of one locus is onlymeasurable in a certain allelic constitution of another or a third orfourth etc. locus.

In a simple theoretical case one could imagine that a homodimeric enzymethat is encoded by a specific gene (1 locus) may be more effective incatalysis if the dimers are slightly different (1 locus but 2 alleles inthe heterozygote) so that, for instance, a more effective catalytic siteis formed. In this case AA′ is superior in catalysis as compared to AAor A′A′. In addition to that, it is well possible that in a biosyntheticpathway this enzyme encoded by the “A” gene (whatever the geneticcomposition may be) could be dependent on the catalysis of anotherenzyme that is upstream or downstream of the particular enzyme in thecascade. It can be easily understood that if the enzyme “A” becomes moreefficient, that this increase in efficiency can only be effectivelyexecuted if there is no limitation in the substrate that “feeds” the “A”encoded enzyme. In the case the substrate used by the A enzyme isprovided by another enzyme (B) whereby the same rule is true(homodimeric enzyme improved by 2 alleles) then improvement is onlyobtained by the combination. In that case AA′/BB′ is better than AA/BB′or AA′/BB and all the other combination where both heterozygous stateswould be absent.

If, on the other hand, the output of the pathway is not limited anymoreby the step that A is controlling, but if an enzyme downstream of Aconstitutes the limiting step, than the effect of different alleles of Ais not measurable and so the locus that is responsible for the enzymedownstream of A is epistatic to A.

A well know example where heterozygote individuals are superior versushomozygous individuals is sickle-cell anemia. Investigation into thepersistence of an allele that is so obviously deleterious in homozygousindividuals led to the finding that the allele confers a small butsignificant resistance to lethal forms of malaria in heterozygousindividuals. Natural selection has resulted in an allele population thatbalances the deleterious effects of the homozygous condition against theresistance to malaria afforded by the heterozygous condition.

It is obvious that superior heterozygosity and epistasis may be presentsimultaneously and the effects described for homodimers can also bevalid for heteromultimers.

In conclusion, this means that the contribution of one particular locuson its own cannot easily be measured or visualized, because at leastpart of the contribution of the individual locus is non-additive andinteracting with the allelic state of one or more other loci. ThereforeQTL mapping of epistatic traits cannot easily be done by traditionalmethods, which are generally assuming additivity between loci.Incorporation of inter-locus-interactions in these methods often resultsin problems with statistical parameter estimation and low power todetect QTLs, due to the high parameterization of the genetic models usedfor this purpose.

Alternative methods trying to solve this problem, are QTL× geneticbackground mapping, which is applied on diallel mating populations(Charcosset A et al. (1994) pp 75-84 and Rebaï A et al. (1994) pp170-177, both in: Biometrics in plant breeding: applications ofmolecular markers; Eds: Ooijen J and Jansen J. CPRO-DLO, Wageningen, TheNetherlands), and QTL× population-mapping, applied on multiple relatedinbred-line crosses (Jannink J-L & Jansen R (2001) Genetics 157:445-454). The latter state that such methods can also be applied toother populations structures.

An interesting population structure for the purpose of detection ofepistatic interactions is the Heterogeneous Inbred Family (HIF) (Haley Set al., (1994) Theor. Appl. Genet. 88, 337-342; Tuinstra M et al.,(1997) Theor. Appl. Genet. 95: 1005-1011), because of its ‘multipleceteris paribus’ property, i.e. the family contains many possiblesub-populations, where in each of them only one QTL is segregating in aspecific homozygous background for the other QTLs.

The construction of HIF-populations is very tedious. It takes severalgenerations of single seed descent, which means it is slow andlabour-demanding. By the time the HIF-population is completed the chosenpopulation parents may not be up to date anymore. Facilities oralternative locations to decrease generation time require highinvestments. Also considering the fact that QTL-alleles of only twoparents are analysed, it is often not worth to invest in suchpopulations for commercial breeding purposes.

A more pragmatic approach for QTL-mapping in the presence of epistasisis presented in U.S. Application No. 2005/0015827. The position andeffect of QTLs in the background given as it is in the ongoing breedingprogram is recurrently monitored. No specific population structure isapplied, as in linkage disequilibrium mapping (see below), and changesin position and effect of QTLs are accepted as a fact of life. The maindisadvantages of this method are the high number of accessions that haveto be analysed and the lack of analytical power to establish specificepistatic effects. In other words, it is not analysed which specificlocus in the genetic background is interacting with the changing QTLs.

A more radical way of avoiding epistatic effects in QTL-analysis is theuse of backcross populations. In this way QTL-effects can be analysed ina more or less constant genetic background, namely that of the recurrentparent. Most backcross population types (for instance backcross inbredlines or BIL's) can be seen as analogues of regular mapping populationtypes where one or more backcrossing generations have been included tocreate a more uniform genetic background, and in several cases, rule outone of the three allelic states of a locus.

In view of the above it is the object of the present invention toprovide a method for mapping traits in organisms, in particular plants,that does not have the above described drawbacks.

SUMMARY OF THE INVENTION

The present invention is based on the finding that it is possible toreadily map the loci that encode complex traits by making use ofgametes, derived from a particular class of abnormal meiotic divisions,and phenotyping the progeny of organisms regenerated from such gametes.

The method of the invention, which is called herein “Reverse ProgenyMapping” or “RPM”, is based on the use of cells, in particular spores,that are formed due to an abnormality in the second division of meiosis,so called Second Division Restitution or SDR and by consequence thesespores are diploid (when the parental plant was also diploid) incontrast to normal spores that are haploid. Such spores are called SDR-0spores and the plants regenerated therefrom are SDR-0 plants.

Second division restitution is just one case of a broader class of saidunreduced spores/gametes. Veilleux R, ((1985) Plant Breeding Reviews 3,253-288), describes the mechanisms by which unreduced gametes are formedand provides a list of the occurrence of unreduced gametes in cropplants. At that time mainly 2 different classes of unreduced gameteswere recognized namely Second Division Restitution (SDR) and FirstDivision Restitution (FDR). Recently, a third class of unreduced gameteshas been published named Indeterminate Meiotic Restitution (IMR) (Lim Ket al. (2001) Theor. Appl. Genet. 103:219-230). For the purpose of thisinvention only SDR is relevant. Another publication that shows that SDRis a natural and widespread phenomenon is Lim K et al. (2004) BreedingScience 54: 13-18.

Due to crossing-over during meiosis I the chromosomes of SDR-0 sporesmay have segments that are heterozygous. In the context of the presentinvention heterozygocity means that the alleles of a gene of the hybridstarting plant are polymorphic, whereas homozygocity means that thealleles of a gene are identical.

When spores produced through SDR are regenerated, diploid plants areobtained with on average a reduced level of heterozygosity as comparedto plants obtained through normal meiosis and selfing (F2 generation).It is estimated that on average SDR events contain 60% homozygocity(starting from a 100% heterozygous hybrid plants) whereas this is 20%for FDR events. The actual level depends on the number and relativeposition to the centromere and of the crossing-overs, which haveoccurred during the specific SDR event.

Only loci that are located on the heterozygous segments in SDR-0 plantscan segregate. Segregation takes place in the next generation, namedSDR-1. The genotype that will produce a specific phenotype in the SDR-1generation will be different from the genotype in the SDR-0 generation.However, segregating phenotypes in the SDR-1 generation can only occurif the SDR-0 plant was to some extent heterozygous. This means that itis sufficient to determine in the SDR-0 generation which loci areheterozygous in order to position the loci that are responsible for thesegregating phenotype in the SDR-1 generation. Identification andlocalisation of the crossing-over breakpoints in the individual SDR-0plants predicts and by consequence maps the position of the locus thatis responsible for the segregation of a particular phenotypic trait inthe SDR-1 progeny.

The present invention thus relates to a method for mapping traits inorganisms, in particular in plants, comprising the steps of:

a) providing a population of SDR-0 organisms, in particular plants, thateach arise from one member of a population of unreduced cells resultingfrom second division restitution, in particular a population ofunreduced spores;

b) producing SDR-1 progeny populations of each of these SDR-0 organisms;

c) phenotyping the SDR-1 progeny populations to identify segregatingtraits within each SDR-1 progeny population;

d) if segregating progeny is present in a SDR-1 progeny populationgenotyping the corresponding SDR-0 organism and comparing the genotypethereof with the genotype of the other SDR-0 organisms to identifyheterozygous chromosomal regions associated with the occurrence of thesegregating trait identified in the said SDR-1 progeny population.

In a specific embodiment, the population of unreduced cells that eachgive rise to a plant of the SDR-0 population is obtained by sorting apopulation of cells, in particular spores, on the basis of size, mass orDNA content and selecting the cells, in particular spores, that have anincreased size, mass or DNA content as members of the population ofunreduced cells, in particular unreduced spores. The cells, inparticular spores, may be sorted by means of a flow cytometer,centrifuge, manually with a micromanipulator or by any other sortingmeans.

Phenotyping the SDR-1 progeny populations can be performed in any wayknown to the person skilled in the art and can in particular be done bymeans of visual observation or by analysis of the content and/orcomposition of ions, transcripts, proteins, metabolites, or combinationsthereof in each SDR-1 organism. Analysing the content and/or compositionof ions, transcripts, proteins, metabolites is for example done withtechnologies such as ionomics, transcriptomics, proteomics, metabolomicsor combinations thereof.

After phenotyping the SDR-1 progeny populations, the SDR-0 organism thatgave rise to SDR-1 progeny populations that segregate are genotyped.Genotyping can be done by any method known to the person skilled in theart. In a preferred embodiment, genotyping of the SDR-0 organisms isperformed by means of a method revealing nucleic acid polymorphisms.Many techniques revealing such polymorphisms are known, such as AFLP,RFLP, SNP, SFP, SSR, RAPD. This list of molecular marker techniques isonly given as an example and is in no way limiting to the invention.

Advantageously, the production of the SDR-1 progeny population isperformed under varying conditions, in particular varying environmentalconditions. The varying environmental conditions are selected fromlaboratory conditions and field conditions. Both types of conditions canfurthermore be varied with respect to weather conditions. This way it ispossible to find and map traits that are phenotypically visible undercertain conditions only.

In a further embodiment the same traits are mapped in different geneticbackgrounds. This way it is possible to find interacting loci in onegenetic background that are not visible in another genetic background.

The invention provides in fact the combination of a novel startingpopulation and known QTL-mapping techniques. Because the level ofheterozygocity in this population is much lower than in otherpopulations the number of organisms that need to be analysed is muchlower than in existent techniques. Moreover, in the most basicembodiment of the invention, only the SDR-0 organism that gives rise toa SDR-1 progeny population that segregates for the trait to be mappedneed be genotyped in comparison to an SDR-0 organism the SDR-1 progenypopulation of which does not segregate for that trait. The locusresponsible for the trait lies then within the heterozygous chromosomefragment present in the SDR-0 organism of the segregating SDR-1 progenypopulation.

Meiotic mechanisms have been described in considerable detail includinga number of aberrant forms, which among others have been termed firstdivision restitution or FDR and second division restitution or SDR. Bothforms of meiosis lead to the formation of diploid gametes due to theabsence of the first or the second meiotic cellular division,respectively. SDR leads to the presence of both sister chromatids in thespores/gametes, which therefore are identical with respect to theirgenetic composition except for those regions, which are heterozygous asa consequence of meiotic recombination (and which were thus alsoheterozygous in the donor-plant, which is the plant donating the SDR-0spores).

In case of a single crossing-over per chromosome arm, the distal end ofthe chromosomes, i.e. from the crossing-over point towards the telomers,will be heterozygous whereas the chromosomal region proximal to thecrossing-over point, which includes the centromere, will be homozygous.

Due to independent chromosome re-assortment during meiosis I, thehomozygous regions of the chromosomes of the SDR events contain geneticinformation derived from either the paternally or maternally inheritedchromosome and therefore an SDR population is very heterogeneous.Nevertheless, an SDR population could be described as a population thatcontains lines that resemble partly heterozygous RILs or HIFs, andpartly heterozygous backcross inbred lines (BILs), the latter withintrogressions in both parent backgrounds.

The occurrence of SDR spores/gametes is in itself well known and isdescribed for several species (see e.g. Ki-Byung Lim et al. (2004)Breeding Science 54: 13-18; Veilleux R. (1985) Plant Breeding Reviews 3,253-288;

Bastiaanssen H. (1997) Marker assisted elucidation of the origin of2N-gametes in diploid potato (PhD thesis) ISBN 90-5485-759-5 (Thisthesis also includes references for many crops)).

Until now complex loci could not or hardly be genetically located bymethods known to the person skilled in the art. The present inventionteaches a completely new method for making mapping populations thatallow scanning the whole genome for loci that segregate. The type oflocus(ci) is (are) not limited to polygenic traits because thesystematics can also be applied for monogenic traits.

The invention provides a novel and surprisingly simple way of analysingloci that may have interdependent and/or epistatic interactions.Moreover, the method is not limited to only 2 loci but can be applied tonumerous loci that interact as long as intra-line segregation can bemeasured or observed.

Inter-line variability is well known to occur between fully homozygouslines (e.g. doubled haploids), whereas intra-line variability refers tothe situation in which a limited number of phenotypic characters differamong individual plants within the line, due to segregation of theremaining heterozygosity in the line parent.

According to the invention it is surprisingly found that plants whichare regenerated from spores that have omitted the second meioticdivision, so called SDR-0 plants, provide unique material for mappingtraits including extremely complex ones, such as those traits that aredependent on the presence of polygenic loci at various allelicconfigurations and effects such as heterosis.

The combination of the identification, enrichment or induction ofunreduced spores of the SDR type, the subsequent regeneration of suchspores into plants (SDR-0) and the molecular characterization of theSDR-0 plants (identification of the residual heterozygous chromosomalsegments) and the correlation/association of such segments with thesegregation for whatever trait or effect in the SDR-1 generation, andthe comparison between different heterozygous SDR-0 lines and theirsegregational pattern, allows to map and identify all loci that eitherdo or do not interact, whether polygenic or not.

Identification and localisation of the crossing-over breakpoints in theindividual SDR-0 plants predicts and by consequence maps the position ofthe locus that is responsible for the segregation of a particularphenotypic trait in the SDR-1 progeny. In addition, fine mappingautomatically takes place dependent on the number of regenerated SDR-0plants and depending on the size of the genetic map.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further illustrated in the non-limitingexamples that follow and that refer to the following figures:

FIGS. 1A and 1B show the occurrence of a normal meiosis for 4 chromosomepairs of a completely heterozygous hybrid and the spontaneous doublingof the chromosomes after the reduction division took place (named“corresponding Doubled Haploids”).

FIG. 2 shows a meiosis of the same heterozygous hybrid as in FIG. 1, butin the situation where the second division fails to take place (i.e. incase of Second Division Restitution).

FIGS. 3A, 3B-1, 3B-2, 3B-3, 3B-4 and 3B-5 show the formation ofspores/gametes that occur in the plant that is regenerated from SDR-0event 3 in FIG. 2.

FIG. 4 shows theoretical individual SDR-0 plants (for one chromosome)that only differ in the extent to which recombination took place.

FIG. 5 shows AFLP patterns of typical F2 lines in cucumber. Everyhorizontal line represents one individual plant. Every vertical columnrepresents a linkage group. Light grey segments represent heterozygousareas, whereas black and dark areas represent respective homozygousareas.

FIG. 6 shows AFLP analysis of typical DH lines in cucumber. Everyhorizontal line represents one individual plant. Every vertical columnrepresents a linkage group. Only black and dark areas are present asexpected in DH's Light grey segments are absent.

FIG. 7 shows the AFLP analysis of typical SDR-0 plants in cucumber.Every horizontal line represents one individual plant. Every verticalcolumn represents a linkage group. Light grey segments representheterozygous areas, whereas black and dark areas represent respectivehomozygous areas. It follows from comparison of these figures that theheterozygosity in these plants is much lower than in an ordinary F2.

FIG. 8 shows the result of an experiment where broccoli microspores froma tetraploid plant (giving diploid (2n) spores) were mixed withmicrospores from a diploid plant (giving haploid (n spores). The largerspores are 2n.

FIGS. 9A and 9B show representative examples of the morphologies ofpollen collected from cold-treated plants (FIG. 9A) versus controlplants (FIG. 9B).

FIG. 10 shows the results of the AFLP analysis on SDR-0 and DH-0 plants.Every individual line represents a single DH-0 plant respectively aSDR-0 plant. Every column represents a linkage group.

DETAILED DESCRIPTION

In greater detail, FIG. 1 depicts the occurrence of a normal meiosis for4 chromosome pairs of a completely heterozygous hybrid and thespontaneous doubling of the chromosomes after the reduction divisiontook place (named “corresponding Doubled Haploids”). In the casedepicted crossing-over has led to the occurrence of 2 parentalchromosomes and 2 recombinants chromosomes per set. Due to thecombination of the individually different homologs from the differentchromosome sets, many different spores/gametes can be produced. In FIG.1 only 3 possibilities are depicted.

Doubled haploids (DH) plants are generated from such “spores”. Theproduction of doubled haploids is a well established technology (Doubledhaploid production in crop plants, Ed: M. Maluszynski, K. Kasha, B.Forster and I. Szarejko. Kluwer Academic publishers,Dordrecht/Boston/London, (2003) ISBN 1-4020-1544-5).

FIG. 2 depicts a meiosis of the same heterozygous hybrid as in FIG. 1,but in the situation where the second division fails to take place (i.e.in case of Second Division Restitution). In this particular case,diploid spores are formed, however in contrast to FIG. 1 where aspontaneous or induced chromosome doubling took place the occurrence ofdiploid spores is caused by the absence of the second meiotic division.In both figures, four chromosome pairs have been depicted and thehomologs are shown in light, respectively dark rod like structures wherethe black circles on the rods represent the centromeres.

The core difference between a doubled haploid plant and diploid SDRplants is clearly seen by the heterozygous segment on the chromosomes inthe SDR plant whereas the DH plant is fully homozygous.

In this theoretical case, the starting plant (donor-plant hybrid AB)that produced the haploid spores from which DHs were made or thatproduced SDR-0 spores, respectively, contains homologous chromosomesthat are completely heterozygous. This means that all alleles of thegenes carried by those chromosomes are different. In practice, however,this is highly unlikely and so this case exemplifies the most extremeheterozygous situation.

From FIG. 2 it is also clear that what determines the ratio ofhomozygous loci versus heterozygous loci in the case of SDR is theextent to which the non-sister chromatids of the homologous chromosomeshave been exchanged due to crossing-over. The limit of cross-over extentfor each chromosomal arm is determined by the position of thecentromere. Of course cross-over from the other end of the chromosomecan also take place and also in this case up to the centromere. Startingfrom plants which are 100% heterozygous, which means all alleles of thegenes carried on the chromosomes are polymorphic, is an extremesituation. In practice this is highly unlikely to occur and thereforethe percentages of heterozygocity of the starting plants will on averagebe lower.

Note also in FIG. 2 the occurrence of SDR-plants that resemble RILs andBILs. In the case of BIL-look-alikes the centromeres are all descendingfrom one and the same original parent, i.e. A or B (see FIG. 1).

Plants regenerated from haploid spores originating from a normal meioticevent in which the chromosome number has been doubled spontaneously orby means of chemicals will be further named DH-0. Plants will be calledSDR-0 in

case the primary regenerant originates from a spore that resulted from ameiotic event that lacked the second meiotic division.

DH-0 plants when self-pollinated will give rise to a progeny (DH-1) thatis genetically 100% identical and is completely fixed in all thealleles. So although the spores (gametes) that are formed on the DH-0plants underwent again meiosis and recombination no geneticrearrangements can take place. This means that this so-called “pureline” is immortalized because no segregation can take place.

Such a pure line can, however, phenotypically show different appearanceswhen grown under different conditions, such as low or high temperatures,or for instance in different climatic zones. The differences that can beobserved are due to environmental variation and valid for all “members”of the line. In other words there will be no “intra-line” variation. Thedifference between plants of different DH1-lines descending fromdifferent DH-0-plants has a genetic basis and is indicated as“inter-line” variation.

In the case of SDR a different picture is observed in the SDR-1generation. FIGS. 3A and 3B depict the formation of spores/gametes thatoccur in the plant that is regenerated from SDR-0 event 3 in FIG. 2. Itis clear from FIG. 3B that by recombination and combination a panoply ofchromosome combinations can occur and obviously the number ofcombinations increases as the number of chromosomes increases. To find a(partially heterozygous) BIL-look-alike for one of both inbred parents,the probability is (½)^(x-1) where x is the number of chromosomes. Tofind a specific (partially heterozygous) BIL-look-alike the probabilityis (½)^(x). The maximum variation that is phenotypically observable isdependent of the extent of heterozygosity in the starting material andalso on the extent of recombination that took place. In the unlikelysituation where no recombination took place at all, or only inhomozygous regions, the SDR-0 regenerant will both genotypically andphenotypically be the equivalent of a doubled haploid (DH-0).

FIG. 4 shows theoretical individual SDR-0 plants (for one chromosome)that only differ in the extent to which recombination took place. Incase segregation for a trait or effect is seen in the SDR-1 generationfor SDR-0 C but not in the A and B case, then it follows that thechromosomal region responsible for the segregation is located betweenthe cross-over positions of B and C. Depending on the availability ofmolecular markers and the number of available SDR-0 plants, loci thatare responsible for segregating phenotypes can thus be very accuratelymapped and associated with known molecular markers.

This method of the present invention is called herein “reverse progenymapping”. The unique feature of this method is that it is usingintra-progeny segregation information of plant progenies from a mappingpopulation of parent plants to perform QTL-mapping. In contrast to theuse of the progeny means, which is often used in traditional mapping,the method makes use of the progeny variation. It is using the contrastbetween the individuals that are heterozygous for a certain chromosomeposition and the individuals that are homozygous for a this position,irrespective for which parental allele. Traditional methods make use ofthe contrast between all three allelic states of a chromosomal position(homozygous parent (AA), heterozygous (AB); homozygous parent (BB)),with an emphasis on the contrast between the two homozygous states.

In another embodiment, reverse progeny mapping can be combined withregular inter-line mapping methods to increase the power ofQTL-detection.

In a further embodiment, individual SDR-1 plant phenotypes can be usedin a general mixture model approach (Jansen R C (1992) Theor. Appl.Genet. 85: 252-260), where the three possible allelic states aremodelled for each SDR-0 individual, that is heterozygous on the analysedchromosome position.

Alternatively, it is possible to use SDR-1 line variance, for example tocalculate an additional likelihood ratio, which can be multiplied withthe regular likelihood ratio, for inter-line mapping, to obtain animproved test statistic.

In another embodiment, it is possible to use simply the score forpresence or absence of segregation in an SDR-1 line. Also in this case,an additional likelihood ratio may be calculated.

The above examples do not exclude other possibilities to combine the useof intra-line and inter-line segregation of the invention with anothertechnique for QTL-mapping.

There are certain conditions in which the method of reverse progenymapping works optimally. In a preferred embodiment the trait, for whichQTLs should be mapped, is segregating in only part of the SDR-1 lines,preferably between 50 and 80%. This means that for certain polygenictraits, for which a higher number of QTLs are segregating in thepopulation, a lower level of heterozygosity is required. If necessarythis can be achieved by further inbreeding in the case of HIFs, or inthe case of SDR, by a second round of SDR, where each SDR-0 individualis used to produce a new SDR-0 plant, resulting in a so called SDR²-0population. Care should be taken that the population size is largeenough to have the whole genome still represented in a heterozygousstate.

According to the present invention SDR-0-based Reverse Progeny Mapping(RPM) combines the ideal characteristics of doubled haploid lines inphenotypic recording and the possibility to assess the effect ofheterozygous loci individually and in interaction with otherheterozygous or homozygous loci.

As already stated earlier, SDR lines are different from DH lines inthose chromosomal regions where they are heterozygous as a consequenceof heterozygosity in the starting material and due to recombination atthose heterozygous segments. This means that for all the other segmentsSDR lines resemble DH-lines. This means that at a phenotypic levelwithin-line segregation is observed for only a limited number ofcharacteristics. Nevertheless, the phenotypic classes which requireheterozygosity of specific loci may be recorded. If, for instance, alocus which determines a specific trait, is heterozygous in the SDR-0generation, it may give a traditional Mendelian segregation of 1:2:1(AA:Aa:aa) in the SDR-1 generation depending on the recombinationposition in the second round of gamete formation. If the Aa phenotype isdifferent from AA and aa, then still it can be recorded.

In the theoretical case where a certain locus has to be in aheterozygous state to be fast growing, SDR-1 lines descending from theSDR-0 plants that have this locus in a heterozygous state, will show a1:1 segregation of faster growth versus normal growth, provided that thesecond round of recombination did not change the recombination positionof the SDR-0 plant. This is a clear example of the application of“intra-line variation” in SDR-0 lines, as opposed to “inter-linevariation” that occurs between DH-1 lines.

Again, the segregation for the theoretical “fast growth” characteristiccan be explained by the presence of heterozygous segments in the SDR-0generation. So it is sufficient to genetically analyse the SDR-0generation to explain and map what phenotypically happens in the SDR-1generation.

The power of the method of the invention is also demonstrated by thefact that interaction between independent loci can be explored. As anexample, a plant is considered in which one locus should be homozygousrecessive (aa) while two other loci should be heterozygous to show thetrait of interest. In this case, no segregation will take place for thetrait of interest if the SDR-0 generation was AA. But if the SDR-0generation was aa then still the trait may segregate for the other loci.Such so-called epistatic effects are difficult to study especially ifthe trait is also dependent on the environment.

The advantage of phenotypic recording of DH populations is nullified bythe lack of segregation within the line, and the lack of heterozygosityof DH-plants. An F2-population will, however, show the desiredsegregation of the trait, but analysis is hampered by the fact that thewhole genome is segregating, providing each of the F2-individuals with adifferent genetic background. This is creating a large statisticalbackground noise which decreases the power of detecting the epistaticQTL-effect. In addition to that, the F2-plant can only be phenotypedonce, which introduces an large environmental error. The possibility toreplicate a DH line in different environments and record the phenotypicdifferences several times, is a big advantage for reliable QTL-mapping.

The same is true with the method of the invention. When sufficient seedsare produced from the SDR-0 lines to allow to test the same SDR-1generation in different conditions, not only loci that contribute in ahomozygous state but also loci that perform different in a heterozygousstate can be recorded. Additionally, recording is possible for epistaticinteractions between loci whether heterozygous or homozygous, and ofcourse for monogenic loci encoding qualitatively or quantitative traits.This is a clear advantage over the existing techniques.

Map distances on chromosomes are expressed in centimorgan (cM) as iswell know to the person skilled in the art. In a 100 centimorganinterval, an average of one crossover occurs per chromatid (Van den BergJ et al. (1997) pp. 334-396 in: Plant molecular biology—a laboratorymanual, Ed. M. Clark, Springer Verlag, Berlin). This means that if oneis looking for mapping a trait which is positioned at 1 cM from thecentromere, there is 1 in 50 recombinants that has a chromatid exchangeup to 1 cM from the centromere. This applies, of course, to either sideof the centromere.

In Table 1 the advantages and disadvantages of using certain populationtypes for QTL-mapping are summarized. Most of them have been mentionedbefore; otherwise they are noted in the table. It is clear from thetable that in comparison with other populations creating SDR-basedpopulations requires limited time and input, with the prospect ofremarkably good results. In this way the SDR-approach combines theadvantages of doubled haploid populations (DH), i.e. quick populationdevelopment at limited cost, with the QTL-mapping potential ofheterogeneous inbred families (HIFs), i.e. reliable phenotyping, strongdetection power of QTLs, including their heterozygous and epistaticeffects, and potential for fine mapping.

TABLE 1 Overview of required efforts to create and use severalpopulation types for QTL- mapping and potential results. EFFORTS RESULTSnumber of population fine mapping markers size (number of labour to timeto required required number of QTL- estimation of recombinations POPULA-produce produce (rough (rough QTL-alleles reliability of detectionQTL-effect for and/or residual analysis of TION TYPE populationpopulation mapping) mapping) per locus phenotype power heterozygoteheterozygosity) epistasis F2 ** ** * * 2 −−/−^(6.8) − = − + BCx***/****³ ***/****³ */**³ **⁵ 2 = + + = − RIL **** **** *** * 2 ++⁷ ++− + ++¹² HIF ***** ***** *** * 2 +⁸ ++ + ++ +++ BIL¹ ***** ***** ** * 2++⁷ ++ +⁹ ++⁹ − DH *** ** * * 2 ++⁷ ++ − − ++¹² SDR *** ** * * 2 +⁸ ++ +++ +++ random² * * ****⁴ *** >=2 ++⁷ = +¹⁰ −/+¹¹ = Efforts (costs/timeframe): *: very limited, **: limited, ***: average, ****: large, *****:very large; Results (except for: estimation of QTL-effect forheterozygote): −−: very poor, −: poor, =: moderate, + = reasonable, ++ =good, +++ = very good; Results (only for: estimation of QTL-effect forheterozygote): −: impossible, =: possible, +: good. BCx: backcrosspopulation after x cyles of backcrossing; RIL: recombinant inbred lines;HIF: heterogeneous inbred families; BIL: backcross inbred lines; DH:doubled haploid lines; SDR: second division restitution derived lines.¹See for instance: Jeuken MJW, Lindhout P (2004): The development oflettuce backcross inbred lines (BILs) for exploitation of the Lactucasaligna (wild lettuce) germplasm; Theor. Appl. Genet. 109(2): 394-401/²Populations of breeding lines, varieties, genebank accessions, used forf.i. LD-mapping ³Effort is increasing with the number of backcrossgenerations (x) ⁴High numbers of markers required for haplotypingmultiple alleles ⁵Higher number required for sufficient genome coverage,especially in higher backcross generations ⁶F3-line phenotyping is morereliable than F2-plant phenotyping ⁷Unlimited replication possible⁸Limited replication possible, depending on seed quantity per line⁹Possible via cross with recurrent parent ¹⁰Only in case of hybrids¹¹Depending on degree of linkage disequilibrium in population ¹²OnlyQTL-interactions between homozygous loci

Zhang X et al. ((2002) Journal of Horticultural Science & Biotechnology78(1), 84-88) found that in pepper the frequency of SDR 2n gametes(pollen) could be increased from <1% to up to 10.5% (average) by 48hours exposure of the plants to 11° C. The maximum SDR occurrence wasmeasured to be 81.3%. This method can be used according to the inventionto increase the number of SDR events and thus the number of SDR-0gametes.

In addition to the spontaneous occurrence of SDR or induction of anincrease in the number of SDR-0 events by environmental stress,different genetic approaches are provided, which allow interference withgene functions involved in the second cell division of meiosis. Suchinterference can either be through mutagenesis or transgenesis.Transgenic approaches aim at the stable or transient introduction of aDNA fragment which modifies the second division of meiosis leading todiploid spores of the SDR type. The modification can occur throughinterference with genetic factors involved in meiotic processes,especially those involved in the second cell division. The interferencecan be established through specific down-regulation of gene expressionbased on post-transcriptional gene silencing (PTGS). PTGS can beachieved through RNA-interference (RNAi) or virus-induced gene silencing(VIGS). The techniques for this are well known in the state of the art.

Yet in another approach, the interference can be established through theover-expression of proteins, which exert a dominant negative effect onthe second division of meiosis leading to SDR.

Thus, in a first embodiment of the invention, the population ofunreduced SDR-0 cells is produced by an organism selected to show anabove-average second division restitution. Alternatively, the populationof SDR-0 cells is produced by an organism that is genetically modifiedto show an above-average second division restitution. The geneticmodification is transient or by stable incorporation into the genome ofa genetic element increasing the number of second division restitutionevents in the organism.

In a still further embodiment, the population of unreduced SDR-0 cellsis produced by an organism that is subjected to environmental stress toshow an above-average second division restitution. Examples ofenvironmental stress are temperature stress, NO₂, nitrous oxide N₂O, orcombinations thereof.

The invention further relates to the use of a mapping populationobtainable by the steps of:

a) providing a population of SDR-0 organisms, in particular plants, thateach arise from one member of a population of unreduced cells resultingfrom second division restitution, in particular a population ofunreduced spores; and

b) producing SDR-1 progeny populations of each of these SDR-0 organisms;for mapping one or more traits in a species.

Irrespective of the approach taken, the target gene needs to be known atthe molecular level. A number of recessive mutants have been describedof potato (pcpc, osos, fcfc) and maize (elongate), which result in anSDR-type of meiosis. The genes, which have been mutated in thesespecific examples, have not yet been identified at the molecular levelbut are excellent candidates to achieve SDR in target species usingmolecular suppression technologies although they are not yet cloned. Thepresent invention relates to the general principle of reverse progenymapping and the fact that not all possible embodiments of inducing SDRin a donor organism have been described is not relevant for theinvention.

Alternatives can be found in genes like DUET (Venkata Reddy et al.(2003) Development 130, 5975-5987) and CYC1;2 (Wang et al. (2004) PlantPhysiology 136, 4127-4135), which have been described in Arabidopsisthaliana and which upon mutation, lead to an aberrant meiosis. Thediploid meiotic products in these mutants are SDR-like and thereforeDUET and CYC1;2 as well as their functional homologues in other plantspecies are candidate target genes to achieve SDR meiosis.

Another candidate target gene is TETRASPORE/STUD (Yang et al. (2003)Plant J. 34, 229-240), which upon knock out leads to absence of celldivision after meiosis. Diploid regenerants of microspores of atetraspore/stud mutant can be SDR-like.

The occurrence of 2n spores or gametes is not restricted to the malegametophyte but there is also evidence that this occurs at the level ofthe female gametophyte. Zagorcheva L (1976), reported the occurrence ofdeviations of macrosporo- and macrogametogenesis in cucumber see:Macrosporgenesis and macrogametogenesis ((1976) Genetics and PlantBreeding 9(5) pp 386-399).

In addition, by making haploids and doubled haploids in cucumberaccording to EP 0 374 755 the inventors found by using AFLP analysis(carried out according to EP 0 534 858) that among the expected doubledhaploids, a certain percentage did not originate from haploid megasporesbut from an unreduced megaspore (2n). This is demonstrated in FIGS. 5, 6and 7.

FIG. 5 shows AFLP patterns of a typical F2 lines in cucumber. Everyhorizontal line represents 1 individual plant. Every vertical columnrepresents a linkage group. Light grey segments represent heterozygousareas, whereas black and dark areas represent respective homozygousareas.

FIG. 6 shows AFLP analysis of typical DH lines in cucumber. Everyhorizontal line represents 1 individual plant. Every vertical columnrepresents a linkage group. Only black and dark areas are present asexpected in DH's Light grey segments are absent.

FIG. 7 shows the AFLP analysis of typical SDR-0 plants in cucumber.Every horizontal line represents 1 individual plant. Every verticalcolumn represents a linkage group. Light grey segments representheterozygous areas, whereas black and dark areas represent respectivehomozygous areas. It follows from comparison of these figures that theheterozygosity in these plants is much lower than in an ordinary F2.

The figures thus show that the originally presumed doubled haploids(FIG. 7) still contain heterozygous sectors, which by definition is notpossible in true doubled haploids (FIG. 6). For comparison FIG. 5 showsthe AFLP analysis of a typical F2 population.

Depending on the amount of polymorphism of the starting material,unreduced spores/gametes and plants thereof may be obtained that containonly one or a very limited number of heterozygous segments. If in suchcase there is a causal relationship between the segregating trait in theSDR-1 generation and the position of the heterozygous segment, in theSDR-0 plant, mapping is very easy and fine mapping can be undertaken inorder to even decrease the size of the heterozygous segment by methodsknown by the person skilled in the art.

In order to obtain SDR gametes, one can use a number of differentapproaches. In many species diploid gametes are produced spontaneously,both on the male and female side, which may be enhanced by specificstress conditions. Regeneration may occur through androgenesis,gynogenesis or parthenogenesis by prickle pollination. Optimisation maybe carried out by pollination using diploid pollen and determination ofthe ploidy level of the offspring.

When SDR meiocytes are produced through male meiosis it is possible toenrich for diploid cells through flow cytometry and fluorescenceactivated cell sorting. Such technologies are per se well known to theperson skilled in the art and have been applied on microspores in thepast (see e.g. Deslauriers C et al. (1991) Biochem. Biophys. Acta 1091,165-172) but these techniques have not yet been used in a mapping methodof the invention.

Unreduced spores or pollen are bigger than their haploid peers.Surprisingly, the mere fact that 2n spores are physically different fromn spores makes it possible through flow cytometry to enrich specificallyfor 2n spores. FIG. 8 shows the result of an experiment where broccolimicrospores from a tetraploid plant (giving diploid (2n) spores) weremixed with microspores from a diploid plant (giving haploid (n spores).The larger spores are 2n.

Thus, according to a further aspect thereof, the invention provides amethod for enrichment of a population of cells, in particular spores orgametes, for SDR cells, in particular SDR spores or gametes, comprisingsorting the population of cells, in particular spores or gametes, on thebasis of size, mass or DNA content and selecting the cells, inparticular spores or gametes, that have an increased size, mass or DNAcontent as unreduced cells, in particular unreduced spores or gametes.In a specific embodiment, the invention provides a method for enrichmentof a population of spores or gametes for SDR spores or gametes, whichmethod comprises sorting the members of the population of spores orgametes by means of a sorting device, in particular by means of FACS.

The invention thus relates to the use of plants and their progenies,regenerated from SDR or SDR-like unreduced gametes for the purpose ofmapping polygenic traits or effects, for the mapping of quantitativetraits or effects, for mapping loci that are interdependent, for mappingloci that show epistatic interactions, for mapping effects as heterosis,respectively combining ability and for mapping of mono- or oligogenictraits.

The present invention has been described herein referring in particularto plants, but the technology is not limited to plants but can also beused for mapping traits in other organisms, such as fungi or animals.Animals may be, for example, fish, birds, reptiles, or mammals.

When the term “unreduced cells” is used in this application “unreducedreproductive cells”, such as spores or gametes, are intended.

The present invention will be further illustrated in the Examples thatfollow and that are not intended to limit the invention in any way.

EXAMPLES Example 1 Production of SDR-0 Organisms in Maize by Means ofIntroduction of Elongate1

Incorporation of nucleic acids in the genome of maize are routineprocedures and methods how to achieve this have been described in e.g.EP-801134, U.S. Pat. No. 5,489,520. EP-97114654.3 teaches Agrobacteriumtransformation of DSM6009 corn protoplasts.

Elongate1 (Barell, P J and Grossniklaus, U. (2005) Plant J. 43,309-320), a nucleic acid sequence that disturbs meiosis resulting in theomission of the second meiotic division was introduced into maize usingthe transformation methods described in the above patent publications.Thus, aberrant spores of the SDR type were obtained. The frequency ofSDR spores that are formed sometimes differed between independenttransformants as a consequence of different genomic sites of integrationof the transgenic nucleic acid sequences.

The microspores or megaspores which were produced as a consequence of anSDR-event contain a diploid set of chromosomes. These diploidmicrospores or megaspores were the starting material for producing SDR-0regenerants. Haploids in maize were routinely obtained from microspores:Pescitelli S and Petolino J (1988) Plant Cell Reports 7: 441-444.Coumans M et al., (1989) Plant Cell Reports 7: 618-621. Pescitelli S etal., (1989) Plant Cell Reports 7: 673-676. Buter B (1997) In vitrohaploid production in maize. In: In Vitro Haploid Production in Higherplants, vol 4, 37-71. Kluwer Academic Publishers. Eds. S Jain, S Sopory& R Veilleux.

Alternatively, haploid maize plants were obtained following natural andartificial pollination with a haploid inducer. In this case seeds wereobtained that contain haploid embryos according to Rotarenco V (2002)Maize Genetics Cooperation News Letter 76: 16.

The above protocols to produce DH maize plants were also applied toproduce SDR-0 maize embryos from SDR-0 cells, of which the formation isinduced by incorporation of Elongate1 into the genome.

In order to obtain the proper balance between the maternal and paternalgenomes at the level of endosperm of the SDR-0 kernels, preferably theinducer line is used as a tetraploid pollinator.

Example 2 Production of SDR-0 Organisms in Maize by Low Temperature orNitrous Oxide Gas Treatment

The frequency of SDR spores was enhanced by treatment of maize plantswith low temperatures or by applying nitrous oxide gas as described byKato, A and Birchler, J A (2006) J. Hered. 1, 39-44.

As a consequence of application of low temperatures or nitrous oxidetreatments, numerous microspores respectively megaspores, were producedwhich are of the SDR-type. The spore population was enriched for thepresence of SDR microspores by using flow cytometry or fluorescenceactivated cell sorting based on the fact that SDR microspores are largerin size as compared to normal haploid microspores. The microspores ormegaspores which were produced as a consequence of an SDR-event containa diploid set of chromosomes. These diploid microspores or megasporesare the starting material for producing SDR-0 regenerants. Haploids inmaize were routinely obtained from microspores as described in Example1.

Haploid maize plants were also obtained following natural and artificialpollination with a so-called haploid inducer. In this case seeds wereobtained that contained haploid embryos according to Rotarenco V (2002)(supra).

The above protocols to produce DH maize plants were applied to produceSDR-0 maize plants from SDR-0 cells, of which the formation is inducedby the treatments specified in this example.

As mentioned in Example 1, preferably use is made of the so-calledhaploid inducer as a tetraploid pollinator in order to balance paternaland maternal genomes at the level of endosperm.

Example 3 Identification and Characterization of SDR-0 Organisms

The SDR-0 maize plants from Examples 1 and 2 can be distinguished fromDH plants (or from FDR (first division restitution) plants) that did notundergo SDR because they are partially heterozygous but have homozygouscentromeric regions. Using the AFLP analysis as described in Example 5for cucumber, DH-0 maize plants that did not undergo SDR will show anAFLP marker pattern without heterozygous areas, while DH maize plantsthat have undergo SDR will show heterozygous areas in the AFLP markerpattern. Subsequently, map construction and statistical analysis wasperformed as described in Example 5 for cucumber.

Example 4 Analysis of SDR-1 Populations and Fine-Mapping of Traits inMaize

The progeny of each of the SDR-0 plants carrying heterozygous regions intheir genome was observed under uniform conditions and segregatingprogenies were classified according to the trait that segregates. TheSDR-0 plants leading to the SDR-1 progenies segregating for a specifictrait are compared with each other and with lines that do not segregatefor that trait. Segregation in the SDR-1 generation may be associatedwith the heterozygous segments of the genome of SDR-0 plants. This wasverified by means of a classical QTL analysis to determine themaximum-likelihood interval between the most flanking markers and thetrait locus. Fine mapping of the locus that is responsible for thesegregation in the SDR-1 generation was performed according to Peleman,J et al., (1995) Genetics 171:1341-1352.

Example 5 Production of and Identification of Heterozygous Segments forMapping in Cucumber SDR-0 Plants

1. Doubled Haploids and SDR-0 Plants

Doubled haploids and SDR-0 plants were regenerated from a F1 derivedfrom a cross between 2 homozygous (pure) cucumber lines. All individualDH and SDR-0 plants were genotypically analysed by means of AFLP.

The production of doubled haploids and SDR-0 plants were carried outaccording to EP 0374 755.

2. AFLP Analysis

AFLP analysis on DH-0 and SDR-0 plants was performed as described by VosP et al., (1995) Nucleic acids Research 23(21): 4407-4414.

The data were processed and analysed with Quantar Pro (Keygene,Wageningen, The Netherlands) allowing codominant scoring of the AFLPmarkers.

3. Map Construction and Statistical Analysis

Genetic maps were calculated using the computer package JoinMap® version2.0 (Stam, P., (1993) Plant J. 3: 739-744).

4. Segregation Characteristics

The following characters where expected to segregate.

Apex splitting

Leaf size

Growth rate

Number of fruits per node

Internode length

Flower size

Fruit size

Fruit colour

5. Results

FIG. 10 shows the results of the AFLP analysis on SDR-0 and DH-0 plants.Every individual line represents a single DH-0 plant respectively aSDR-0 plant. Every column represents a linkage group. A clearly distinctclassification can be made between DH lines and lines carrying aheterozygous segment (light grey areas). Segregation in the SDR-1generation of the above mentioned traits is associated with theheterozygous segments accordingly.

6. Fine Mapping

Fine mapping of the locus that is responsible for the segregation in theSDR-1 generation is performed according to Peleman J et al., (2005)Genetics 171: 1341-1352.

Example 6 Enhancement of the Formation of Unreduced Spores/Gametes inSweet Pepper (Capsicum annuum L.)

In order to increase the frequency of unreduced spore/gamete formation,cold stress was applied as an inducer, exactly as described by Zhang Xet al. (2002, supra).

For this purpose, flowering plants of sweet pepper containingpre-meiotic floral buds and growing at 23° C. were exposed for 2 days to11° C. After this cold shock, the buds were harvested and pollen wereextracted by opening the anthers using dissecting forceps and scalpel.The pollen were subsequently transferred on a microscopic glass slideand stained for viability using a drop of aceto-carmine. Cover slideswere put on top of the suspension which was investigated usinglight-microscopy.

As a control, pollen was collected form sweet pepper plants which weregrown at 23° C. FIG. 9 shows representative examples of the morphologiesof the pollen collected from the cold-treated plants (FIG. 9A) versusthe control plants (FIG. 9B). As can be seen, the number of pollen witha larger size indicative for being derived from unreduced spores isstrongly increased for the cold-treated plant. In this particularexample it was estimated that the % of enlarged spores mounted up to 25due to the cold treatment. As such the enhancement of the formation ofunreduced spores by temperature stress is shown to be highly feasible.

Various modifications and variations of the described products andmethods of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in chemistry, biology orrelated fields are intended to be within the scope of the followingclaims.

1. A method for enriching a population of unreduced second divisionrestitution (SDR)-0 cells comprising sorting the population of cells onthe basis of size, mass or DNA content and selecting the unreduced SDR-0cells that have an increased size, mass, or DNA content as members ofthe population of unreduced SDR-0 cells, wherein SDR-0 cells areenriched in the population of cells.
 2. The method of claim 1, whereinsorting the population is performed by means of a fluorescence-activatedcell sorter.
 3. The method as claimed in claim 1, wherein the cells arespores or gametes.
 4. The method of claim 1, wherein the unreduced SDR-0cells are sorted by means of a flow cytometer, centrifuge, or manuallywith a micromanipulator.